Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens

doi:10.3969/j.issn.1004-1524.20230300

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (3): 503-514.DOI: 10.3969/j.issn.1004-1524.20230300

• Animal Science • Previous Articles Next Articles

Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens

LI Tian’en¹^,²(), ZHOU Sihan¹^,², SUN Hongchao², FU Yuan², SHI Tuanyuan²^,^*(), YAN Wenchao¹^,^*()

1. College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023, Henan, China
2. Institute of Animal Husbandry and Veterinary Medicine, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2023-03-10 Online:2024-03-25 Published:2024-04-09

Abstract

Abstract:

Eimeria tenella is a highly pathogenic protozoan that seriously endangers the healthy development of the chicken industry. Dense granule proteins (GRAs) are important intracellular parasitic functional proteins of the apicomplexan protozoa, which have high immunological application value. However, there is no exact report on the dense granule proteins of coccidia. In order to explore the GRAs of Eimeria tenella and study their functions, two hypothetical dense granule proteins (hEtGRA), hEtGRA12 and hEtGRA9, were identified from E. tenella Beijing strain by bioinformatics, molecular biology and immunology. The purified proteins were used to immunize mice to prepare polyclonal antibodies, and the antigenicity of the two proteins was detected by ELISA and Western blot. The results of molecular cloning showed that the coding regions of hEtGRA12 and hEtGRA9 genes were 1 188 bp and 1 110 bp in length, encoding 395 and 369 amino acids, respectively. The similarity of hEtGRA12 and hEtGRA9 with GRA12 and GRA9 of other apicomplexan protozoa was 28.8%-39.6% and 27.5%-29.5%, respectively. SDS-PAGE showed that the recombinant proteins rhEtGRA12 and rhEtGRA9 were 63.6 ku and 67.0 ku, respectively. The results of ELISA and Western blot showed that the recombinant proteins rhEtGRA12 and rhEtGRA9 could induce high antibody levels in mice, and could be recognized by chicken anti-E. tenella positive serum, indicating good antigenicity. In this study, two hypothetical dense granule protein genes hEtGRA12 and hEtGRA9 of Eimeria were identified, and recombinant proteins rhEtGRA12 and rhEtGRA9 were obtained, which laid a foundation for the study of gene function and immune application of dense granule protein of Eimeria.

Key words: Eimeria tenella, hypothetical dense granule protein, prokaryotic expression, polyclonal antibody, antigenicity

CLC Number:

S858.31

LI Tian’en, ZHOU Sihan, SUN Hongchao, FU Yuan, SHI Tuanyuan, YAN Wenchao. Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens[J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 503-514.

Figures/Tables 9

Table 1 Primers for cloning dense granule protein hypothetical genes in E. tenella

假定致密颗粒蛋白基因 Hypothetical dense granule protein gene	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
ETH_00023950	ATGGGATTGACTGGCTTTT	TCATGAGTCTCCCTTACTC
ETH_00031740	ATGGCGCCCCGGCTTTCC	CTAGGCCGCCTCCTCCTCGTTGTCC
ETH_00024035	ATGCGATCCTCGCAGCGT	TTAATTTCTCTTCGAGGCCG
ETH_00028475	ATGCCCCACCACGTGCTGG	TCAGCAGAGAAAGGCTGCATAGC
ETH_00009660	ATGGTATATCTGTTATTGCC	CTAGCTAGCCAAGTAGGCACTTGCC
ETH_00025090	ATGCCTGCAGCTGCAGCTCCC	TCAAAACTGCATCTCCAGCAGCAGC
ETH_00023930	ATGGTTGTGTCGTTGATTGTGTGTG	TTAACTTTGCGCCCTCTCTGCTTCA
ETH_00035235	ATGAATGAAGTCGAAGATGTCC	TCAGCACAGCAGGCCGCAGCCCACC
ETH_00030725	ATGCCCTTAAAGATGGCTTTGC	TTAGTCATTTGTTGCTGTAGCAACA
ETH_00010075	ATGCGGCGTCTGCAGCTGAACT	TGTTGCTGCTTCTGCTGCTGCTGCT
ETH_00032355	ATGAAAATGAAAAAAGAAAATC	TCATTCATCTCCAATCCACATCCGC

Fig.1 Cloning of hEtGRA12 gene and hEtGRA9 gene M, DNA maker; 1, hEtGRA12 gene fragment; 2, hEtGRA9 gene fragment.

Fig.2 Homology and phylogenetic analysis of hEtGRA12 protein A, Analysis of homolog; B, Analysis of sequence similarity; C, Analysis of kinship, △ represented sequence of hEtGRA12 protein.

Fig.3 Sequence similarity analysis of hEtGRA9 protein A, Sequence similarity analysis of hEtGRA9 and TgGRA9 protein; B, Sequence similarity analysis of hEtGRA9 and BbGRA9 protein. ●,Sequence of hEtGRA9 protein; ▲, Sequence of TgGRA9 protein; ◆, Sequence of BbGRA9 protein.

Fig.4 Bioinformatics analysis of EtGRA12 protein A, Prediction of signal peptide; B, Prediction of transmembrane region; C, Analysis of hydrophilic/hydrophobic; D, Prediction of secondary structure, blue represented α-helix, purple represented random coi, red represented extended strand; E, Prediction of antigen epitope. The same as in figure 5.

Fig.5 Bioinformatics analysis of hEtGRA9 protein

Fig.6 Conservative analysis of protein functional domain Green, Signal peptide; Blue, α-Helix; Purple, Random coil; Red, Extended strand.

Fig.7 Recombinant expression of hEtGRA12 and hEtGRA9 genes A, Identification of pET32a (+)-hEtGRA12 plasmid by double enzyme digestion; B, Expression and purification of hEtGRA12 protein; C, Identification of pET32a (+)-hEtGRA9 plasmid by double enzyme digestion; D, Expression and purification of hEtGRA9 protein; M1, DNA maker; M2, Protein maker; 1, pET32a (+)-hEtGRA12 plasmid; 2, Digestion product of pET32a(+)-hEtGRA12 plasmid; 3, Unpurified hEtGRA12 protein; 4-5, Purified hEtGRA12 protein; 6, pET32a(+)-hEtGRA9 plasmid; 7, Digestion product of pET32a(+)-hEtGRA9 plasmid; 8, Unpurified hEtGRA9 protein; 9, Purified hEtGRA9 protein.

Fig.8 Antigenicity analysis of rhEtGRA12 and rhEtGRA9 A, Western blot analysis of recombinant protein rhEtGRA12; B, Western blot analysis of recombinant protein rhEtGRA9; M, Protein marker; 1 and 4, Negative chicken serum; 2 and 3, Anti-Eimeria tenella positive chicken serum; C, Detection of mouse anti-rhEtGRA12 and mouse anti-rhEtGRA9 polyclonal antibody titers.

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Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens

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